The goal of this project is to re-invent concepts regarding the regulation, targeting, and particularly the translational use of protein kinase G (PKG) activation for the goal of treating myocardial disease. PKG is the primary enzyme activated by the second messenger cyclic GMP (cGMP) and a prominent regulator of vascular tone. Its role in the cardiomyocyte has been more controversial, but growing evidence shows that stimulating PKG provides a potent anti-stress and pathophysiological brake, countering pro- hypertrophic/fibrotic signaling, mechano-sensing and arrhythmia, and improving diastolic function. New data from our lab now shows it can also profoundly impact microRNA formation, protein quality control, and autophagy. Cyclic GMP is generated by either a nitric oxide or natriuretic peptide signaling-pathway. While both have long been viewed as interchangeable, our recent work shows prominent differences in their role and regulation in the cardiomyocyte. They operate in nano-domains regulated by specific phospho- diesterases; notably PDE5 and PDE9 that target NO and NP stimulated cGMP, respectively. Thus, how one activates PK effectively depends upon the disease condition and thus how cGMP is being generated, which PDEs are involved, and even the post-translational state of PKG. For example, oxidative stress, which depresses NO-stimulated cGMP also oxidizes PKG, which we showed reduces its protective effects while also altering its response to agonists. Estrogen depletion impairs NO-stimulated cGMP in females, compromising PKG activation strategies dependent on this pathway. This R35 program develops four innovative research programs aimed at ultimately improving our therapeutic use of PKG activation: 1) Dissect nano-domain controls, defining protein partners, selective PKG kinase targets, their dynamics in varying diseases, and how they can be more effectively regulated; 2) Identify how co-morbidities such as obesity, metabolic syndrome, and post- menopause limit PKG activation strategies, and develop methods to circumvent them; 3) Discover novel signaling by which PKG activation provides benefit, including new roles in autophagy, proteosome trafficking, pathological mechanosensing and transcriptional controls; and 4) Develop a PKG proteotype using new proteomic methods applied to human heart biopsies and blood. Through this work, we aim to transform concepts of PKG therapy for heart disease, based on its biology to derive an effective personalized approach.